Abstract RNA modifications are found in rRNA, tRNA, mRNA, snRNA and miRNA in all three primary phylogenetic domains. Recent technological advances that allowed mapping of selected RNA modifications on a transcript-wide scale revealed wide-spread distribution of N6-methyladenosine (m6A), pseudouridin (?), N1-methyladenosine (m1A), 5-methylcytosine (m5C), ribose 2?-O- methylation (Nm) on mRNA. For example, there were over 62,000 m6A peaks in over 10,000 genes in mice and over 118,000 m6A peaks in over 12,000 genes in the human transcriptome. The focus of the phase I project will be on m6A although the technologies that will be developed could be easily adapted to other RNA modifications. The main function of m6A is in regulation and distributing transcripts into either RNA decay, RNA spicing, or translation pathways including both promotion and inhibition of translation. It has been demonstrated that m6A RNA modification controls a plethora of systems, including stem cell proliferation and differentiation, cellular heat shock responses, spermatogonia differentiation, maternal-to-zygotic transition, X-chromosome inactivation, UV DNA damage response, neurogenesis and tumorigenesis. Despite widespread recognition of important functions of RNA methylation, the technologies to monitor and image methylation at specific sites of RNA are lacking. Novel tools for site-specific detection of RNA methylation in cells and tissues are urgently needed. The goal of this project is to develop novel fluorescence-based methods and reagents for sequence specific imaging of RNA methylation. Our novel imaging reagents will produce a greatly enhanced fluorescence signal only when a methylated base is present in the vicinity of a specific RNA sequence which will be very beneficial for background-free imaging. Additionally, we propose to develop novel probe(s) designed to suppress the background signal from nontarget RNAs, especially rRNA. These reagents when used together will allow imaging of even low abundance methylated RNAs. In Aim 1, we will use model RNA targets to develop and characterize 3 variants of reagents for sequence-specific imaging of RNA methylation which will be tested in Aim 2 for imaging methylated RNA in cells to validate their practical utility and commercialization potential. At the end of this Phase I project, we will establish a solid foundation of the Phase II project where we will expand the use of our imaging reagents to sequence specific imaging of RNA modifications in different types of tissues. The future products will be packaged as highly validated assay kits that will be suitable for most types of RNA modifications, in different cell types and different tissue samples. Our method could become a standard approach for analyzing RNA modifications in any sample and will enable researchers to perform immunofluorescence and immunohistochemistry for RNA modifications in important tissue samples for the first time, including brain and cancer tissues. It may further serve as an important diagnostic or prognostic clinical tool for detecting altered methylation levels in the mRNA of tumors.